A Novel Nanopore-Based Genotyping System for Norovirus GII: Validation and Application to Pediatric Gastroenteritis Cases in Moscow, Russia

Norovirus is a leading cause of acute gastroenteritis, with genogroup II (NoV-GII) being predominant. This study presents a novel genotyping system for norovirus GII, combining long-range PCR to amplify the complete ~3.2 kb RdRp-VP1 region with Nanopore sequencing and a dedicated bioinformatics pipeline. This approach provides comprehensive, high-resolution genomic data, representing a significant advancement over conventional methods. Encompassing the recombination site and the full lengths of both genotyping regions in a single amplicon enables sensitive detection of mixed infections and recombinant variants, even at concentrations as low as 1% for minor genotypes. We validated the system through retrospective analysis of 115 pediatric acute gastroenteritis cases in Moscow, Russia (2021-2025) with a high viral load of NoV-GII, selected from 3061 screened stool samples. The analysis revealed the predominant circulation of GII.4[P16] norovirus strains, while GII.17[P17] emerged as the second most prevalent genotype after 2021. In contrast, previously common genotypes GII.4[P31] and GII.3[P12] sharply declined. Mixed infections were found in 4% of cases. With the data obtained, we are doubling the number of long (>3000 bp) NoV-GII genomic sequences from Russia available in public databases and providing unique surveillance data for the Moscow region covering the last five years. The results establish the system as a robust framework for high-resolution surveillance, supporting timely detection of emerging strains and informed public health response.